African trypanosomes regulate mitochondrial assembly during their life-cycle. Within the mammalian bloodstream trypanosomes repress mitochondrial activities, lack detectable cytochromes and rely on glycolysis for ATP production. In the insect vector, trypanosomes develop an electron transport chain and oxidative phosphorylation. The mechanisms controlling the expression of mitochondrial genes and nuclear genes encoding mitochondrial proteins and RNAs are among the major questions to be addressed in this proposal. Developmental Regulation of Nuclear Encoded Mitochondrial Proteins. Cytochomes c and c1 are abundant in insect stage trypanosomes and undetectable in bloodstream forms. Surprisingly, their abundance is post- translationally regulated. The kinetics of turnover suggests that these proteins are targeted for degradation in the bolldstream trypanosomes prior to import into the mitochondrion. Our studies will focus on elucidation of the import pathways for nuclear encoded mitochondrial proteins and the mechanism of developmentally regulated protein import. Mechanism of RNA Import into Trypanosome Mitochondria. All of the mitochondrial tRNAs in T. bucei are nuclear encoded and therefore must be imported for the production of functional mitochondria. The mechanism of tRNA import will be studied using an in vitro import system we have recently developed. This invitro system will be used to determine the features of the tRNAs necessary for import and the mitochondrial proteins and cytosolic factors involved. Devleopmental Regulation of RNA Editing. RNA editing is fundamentally important in trypanosome mitochondrial assembly since it is required for the formation of complete open reading frames for several mitochondrially encoded proteins. We will purify the RNA ligase and terminal uridylyl transferase (TUTase) from RNA editing complexes and establish their funtion both in vitro and in vivo. In addition, developmentally regulated proteins will be identified and the role of these proteins in selective editing of mRNAs in different liffe- cycle stages will be determined. Taken together these studies will provide us with a better understanding of biochemical processes involved in the control of trypanosome mitochondrial biogenesis.